Regulation of the DEAH/RHA helicase Prp43 by the G-patch factor Pfa1

Significance DEAH/RHA helicases remodel RNP complexes in central processes of RNA metabolism such as transcription, splicing, or translation at the cost of adenosine triphosphate (ATP) hydrolysis. These enzymes require activation to function efficiently in their specific cellular context. We show that G-patch factors do not only recruit the DEAH/RHA helicase Prp43 to its cellular localization, but also interfere with its mechanism of motion along RNA substrates. Instead of dissociating from the substrate after a single round of catalysis, Prp43 bound to G-patch translocates continuously along the RNA, resolving stable RNA structures. Our research reveals the mechanism of RNA duplex unwinding by Prp43 and demonstrates how this process is regulated by its cellular cofactors.


Fluorescence-Labeling of ctPrp43
To generate two maleimide-reactive fluorescence-labeling sites in ctPrp43, eight native cysteine residues had to be considered. C148, C214, and C377 are buried inside the protein and inaccessible to the coupling group. C303, C323, C441, C508, and C543 are surface exposed and therefore accessible to the fluorescence dye. C303 is located in the RecA2 domain and remained unchanged while the other exposed cysteines were replaced by site directed mutagenesis (C323V, C441A, C508A and C543S). The second labeling site was generated by introducing a cysteine residue at position K170 in the RecA1 domain. The resulting variant is denominated Prp43Cys. A control mutant denominated Prp43Cys2 was generated, where the labeling site in RecA1 was introduced at position S187. The mutant proteins were expressed and purified as described above with the addition of 5 mM DTT to the lysis buffer and 1 mM TCEP to the His-trap loading and elution buffers. The purified proteins were mixed with Cy3-maleimeide and Cy5-maleimide (Cytiva) dissolved in dimethylsulfoxide at a molar ratio of 1:2:3 (protein:Cy3:Cy5) and incubated for 10 min at 20°C. Excess dye was removed by Ni-sepharose affinity chromatography, labeled proteins were eluted in 50 mM Tris/HCl (pH 7.5), 400 mM NaCl, 5% (v/v) glycerol, 2 mM MgCl2, 250 mM imidazole. The labeled proteins were dialyzed twice against 50 mM Tris-HCl, pH 7.5, 300 mM KCl, 3 mM MgCl2 using Slide-A Lyzer Dialsis Casette G2 3.5K (Thermo) for 1 h at 4°C. The labeled proteins were concentrated to final concentrations between 40 and 70 µM (Amicon Ultra 50K, Millipore).
To determine the degree of labeling, i.e. the average number of fluorophore molecules per molecule Prp43, the absorption of the labeled protein was measured at 280 nm as well as at the absorption maxima of Cy3 and Cy5, 552 nm and 650 nm. Both Cy3 and Cy5 also show absorption at 280 nm, thereby increasing the A280 for the labeled protein. The correction factors (CF) required to eliminate the contribution of the dyes at 280 nm were provided by the manufacturer as CFCy3=0.08 and CFCy5=0.05 (Cytiva). The dye to protein ratio was then calculated for both dyes using where Axxx is the absorption of labeled Prp43 at the specified wavelength, Ɛ280=72700 M -1 cm -1 is the extinction coefficient of ctPrp43 at 280 nm (ProtParam (4)), and Ɛ552=150000 M -1 cm -1 and Ɛ650=250000 M -1 cm -1 are the extinction coefficients of Cy3 and Cy5 at their absorption maxima. Prp43Cys was labeled by 77% with Cy3 and by 66% with Cy5. Prp43Cys2 was labeled by 80% with Cy3 and by 81% with Cy5.

Sample preparation for TIRF microscopy
Cover slips and objective slides were cleaned by bath sonication in 1 M KOH and exposure to plasma (FEMTO plasma cleaner, Diener Electronic GmbH, Germany). Surfaces were then silanized by sonication in 3.9 mM N1- [3-(trimethoxysilyl)  Biotin-PEG-functionalized cover slips were incubated for 5 min at room temperature with TIRF buffer B containing additionally 10 mg ml −1 BSA and 1 μM neutravidin (Thermo Scientific). Excess neutravidin was removed by washing with the same buffer containing 1 mg ml −1 BSA. The labeled RNA was applied to the surface and images were recorded after the addition of TIRF buffer C. To observe binding of Prp43 to the RNA, the buffer was supplemented with 5 µM scPrp43 or 0.5 µM scPrp43 and 2.5 µM scPfa1(gp). The influence of nucleotides was studied by adding 2 mM ADP, AMPPNP or ATP with an energy recycling system (see above) to TIRF buffer C.

TIRF microscopy
TIRF imaging was performed on an IX 81 inverted microscope using a PLAPON 60 × 1.45 numerical aperture objective (Olympus, Japan). Fluorescence was excited by a 561 nm solidstate laser operated at a power of 25 mW. Images were recorded with an electron multiplying CCD (charge-coupled device) camera (CCD-C9100-13, Hamamatsu, Japan). In FRET experiments, color channels were separated by projecting donor and acceptor emission on different parts of the CCD chip using an image splitter (dual view micro imager DV2, Photometrics, USA), filter specifications HQ 605/40, HQ 680/30 (Chroma Technology).
Movies were recorded at a rate of 30 frames per second. The experiments were carried out at 22 °C.

Data analysis
Fluorescence time courses for donor (Cy3) and acceptor (Cy5) were extracted using custommade Matlab (MathWorks) software as described (5,6). A semi-automated algorithm (Matlab) was used to select anti-correlated fluorescence traces (correlation coefficient <0.1) exhibiting characteristic single fluorophore intensities. The bleed-through of Cy3 signal into the Cy5 channel was corrected using an experimentally determined coefficient (∼0.13 in our setup; (5)). Leakage of Cy5 fluorescence into the Cy3 channel was not detected. All trajectories were smoothed over three data points and truncated to remove photobleaching and photoblinking events. Traces with lifetimes of Cy3 or Cy5 less than 20 frames (0.66 s) or with multiple photobleaching steps were excluded from the analysis. The FRET efficiency was defined as the ratio of the measured emission intensities, Cy5/(Cy3+Cy5) (6). FRET time courses were fitted by Hidden Markov modeling using the vbFRET software package (http://vbfret.sourceforge.net/) (7). Models with different number of states were considered for each data set. FRET changes of <0.1 in idealized trajectories were not considered as transitions. Transitions lasting for only one frame were not included in the analysis as well. About 5% of all traces were poorly idealized by Hidden Markov modelling and eliminated from subsequent analysis. Two-dimensional contour plots were generated from time-resolved FRET trajectories. The set of all FRET traces for a given condition was compiled in a histogram, which was fitted to a sum of Gaussian functions using Matlab code (5). Mean FRET values (mean±sd) and population distribution (p=area under the curve±sd) were calculated from three independent datasets and are summarized in Table 1 and Table 3. Dwell times of different FRET states of fluctuating traces were extracted from idealized trajectories. The dwell time histogram for each transition was fitted to an exponential function, y=y0+Ae −t/τ . Rates (k) were calculated by taking the inverse of dwell times (τ).

Isothermal titration calorimetry (ITC)
The binding affinity of ctPfa1(gp) to ctPrp43 was measured by ITC with a MicroCal VP-ITC (Malvern Panalytical) using a concentration of 4 μM Prp43 in the cell and 53 µM Pfa1(gp) in the syringe. The reaction buffer contained 20 mM Tris/HCl (pH 7.5), 200 mM NaCl, 5% (v/v) glycerol and 2 mM MgCl2. The initial injection of 5 µl was followed by 15 µl injections performed at a speed of 0.5 μls -1 with intervals of 360 s between injections. The reference energy was set to 10 µCals -1 and the binding was monitored at 25 °C. Stoichiometry of binding and dissociation constant were determined in three independent experiments, data analysis was carried out using MicroCal VP-ITC Analysis software (Malvern Panalytical).

RNA binding assay
The RNA binding of Prp43 was measured by fluorescence polarization spectroscopy using a VICTOR Nivo Multimode Microplate Reader (PerkinElmer). The binding of 6 nM 3′ 6carboxyfluorescein-labeled A20-RNA (Sigma Aldrich) to up to 100 μM scPrp43 was monitored as triplicates at room temperature in 20 mM Tris/HCl (pH 7.5), 200 mM NaCl, 5% glycerol and 3 mM MgCl2. For measurements in presence of Pfa(gp), the complex was formed by adding a 5-fold molar excess over Prp43 and incubating for 10 min at room temperature. Experiments in presence of ADP or AMPPNP were performed at a constant concentration of 3.5 mM throughout all measurements. The excitation wavelength was 480 nm and the emission was detected at 530 nm for 500 ms. The data were normalized by setting the maximum of measured polarization to 100% and the polarization measured without the addition of protein to 0%. The data were fitted by nonlinear regression with a sigmoidal dose response equation where r is the measured polarization, r0 the initial polarization, Δrmax the maximum amplitude of polarization, [E]T the total protein concentration and KD the dissociation constant, using the analysis software OriginPro 9.1.

ADP binding assay
Binding of ADP to Prp43 was measured by fluorescence polarization spectroscopy using a Fluorescence spectrophotometer FluoroMax 3 (Horiba Scientific). The binding of 100 nM mant-labeled ADP (Jena Bioscience) to up to 20 μM ctPrp43 was monitored as triplicates at room temperature in 20 mM Tris/HCl (pH 7.5), 200 mM NaCl, 5% glycerol and 3 mM MgCl2. For measurements in presence of Pfa(gp) and ssRNA the complex was formed by adding a 5-fold molar excess over Prp43 and incubating for 10 min at room temperature. The excitation wavelength was 360 nm and the emission was detected at 450 nm. The data were fitted by nonlinear regression as detailed above.
ADP release assay ADP release from ctPrp43 was followed through FRET, with Prp43's tryptophan residues acting as the FRET donor, and the mant-label on the nucleotide as the FRET acceptor. For the determination of dissociation rates (koff), 10 μM of labelled nucleotide was preincubated for 10min at room temperature with 1 μM ctPrp43 in 20 mM Tris/HCl (pH 7.5), 200 mM NaCl, 5% glycerol and 3 mM MgCl2, in presence or absence of a 5-fold molar excess of ctPfa1(gp) or A20 RNA. The sample was rapidly mixed with 1 mM unlabeled "dark" ADP using a Stopped Flow Spectrometer SX20 (Applied Photophysics). The excitation wavelength was set to 280 nm and the emission of the mant-label was detected at 450 nm using a 395 nm filter to eliminate stray light. The reaction was followed for 60 to 1800 s and the time curves were fitted to a one phase exponential function using the analysis software OriginPro 9.1. The length of the lag phase was defined as the time between the start of the experiment (t0) and the minimum fluorescence value preceding the major fluorescence increase of the time course.

Supplementary Figures
Supplementary Figure S1: ATPase activity and binding affinity of the Prp43-Pfa1(gp) complex. a SDS-PAGE assessing the purity of ctPrp43, scPrp43 (wt and variants, 10 % acrylamide gel, above), ctPfa1(gp) and scPfa1(gp) (17.5 % acrylamide gel, below). A protein ladder ranging from 10-180 kDa (Thermo) is shown as reference. b Steady-state ATP hydrolysis rates of scPrp43, ctPrp43 with and without His-tag, mutant ctPrp43 (Prp43Cys and Prp43Cys2) with reactive cysteines in RecA1 and RecA2, and Cy3/Cy5 labeled Prp43Cys and Prp43Cys2 at 2 mM ATP. The labeling efficiency of Prp43Cys was 77% and 66% for Cy3 and Cy5, respectively. Prp43Cys2 was labeled by 80% with Cy3 and by 81% with Cy5. Pfa1(gp) and A20-ssRNA were added at saturation. kcat±sd were obtained in N=3 independent measurements. b mant-ADP binding affinity to Prp43 determined by fluorescence polarization spectroscopy in the presence of Pfa1(gp) (red) or Pfa1(gp) and ssRNA (purple). Shown are mean values; error bars correspond to the sd derived from N=3 independent measurements. The table indicates affinity constants (KD(mant-ADP)±sd). In presence of Pfa1(gp) and ssRNA less than 50% polarization was reached, the indicated KD is an estimate of the lower limit.